Integration of a DNA copy of the retroviral genome into a chromosome of the host cell is an essential step in the retroviral replication cycle. The objectives of this project are to understand the detailed molecular mechanism of the integration reaction and to facilitate the development of inhibitors that block this step in the replication cycle. We have previously shown that the viral integrase protein carries out the central steps of the integration reaction in vitro. Our recent work has continued to focus on the biochemical activities of the HIV integrase protein. Integrase catalyzes two distinct reactions: site-specific cleavage of two nucleotides from the 3' ends of the viral DNA and a subsequent reaction that inserts the resulting processed ends into a target DNA. Stereochemical analysis of these reactions supports the view that they both proceed by a one-step mechanism, not involving a covalent intermediate between integrase and the DNA substrate. We have analyzed the functional organization of HIV integrase by expressing and purifying mutant proteins with changes at selected amino acid positions, or deletions extending from the N- or C-terminus. Substitution of conserved amino acids in a central part of the protein that is highly conserved among retroviral integrases abolished catalytic activity, suggesting a key role for this part of the protein in catalysis. In contrast, a conserved motif near the N-terminus is not essential for catalysis, but may be important for protein-DNA or protein-protein interactions. Retroviral DNA made by reverse transcription after infection of a sensitive cell exists as part of a large nucleoprotein complex, derived from the viral core. Although purified integrase protein carries out the DNA cutting and joining steps of integration in vitro, some aspects of the reaction are not efficiently reproduced with integrase alone, but are reproduced when in vitro reactions are carried out with complexes isolated from infected cells. We are analyzing such complexes, isolated from cells infected with Moloney murine leukemia virus, to determine the factors that contribute to this grater fidelity.